The goal of this proposal is to elucidate the pathway of programmed cell death by a novel antifungal agent and explore its use as an antimycotic adjunct. Amiodarone is an effective antiarrhythmic drug that was recently discovered to have potent and broad range fungicidal activity. We have shown that amiodarone toxicity in the yeast Saccharomyces cerevisiae is mediated by disruption of calcium homeostasis, followed by the appearance of apoptosis markers and cell death. In Aim 1, we propose to use a combination of biochemical and cell biological approaches to determine the identity and temporal order of events leading from the initial burst of cytosolic calcium to cell death. We will seek to validate key findings in the pathogenic yeast Candida albicans as proof-of-principle for the universality of the fungicidal mechanism of amiodarone. In Aim 2, we will use a variety of genome-wide approaches to identify the genes and signaling pathways that contribute to amiodarone-induced cell death. Genes identified by these studies will be organized into biomodules and placed in cellular pathways by integrating experimental data from high-throughput biochemical assays with information from online databases to give a global view of drug toxicity. We have shown that low doses of amiodarone exhibit potent synergism with existing antifungals against pathogenic fungal species of Candida, Cryptococcus and Aspergillus. Drug synergy from these in vitro studies will form the basis for combination therapy that will be explored in a murine model of systemic Candidiasis (Aim3). Taken together these studies will develop the calcium-mediated cell death pathway as a major new drug discovery target opportunity. Amiodarone will serve as a model test compound for targeting this pathway and for validating the potential of a new class of antifungal potentiating agents. The public health relevance of this project arises from the emergence of new fungal pathogens and drug resistant fungi, and the urgent need for alternative strategies in the management of fungal infections.